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Edited by:
Eugenio Benvenuto,
ENEA, Italian National Agency for New
Technologies, Energy and Sustainable
Economic Development, Italy
Reviewed by:
Biswapriya Biswavas Misra,
University of Florida, USA
Felix Stehle,
Technical University of Dortmund,
Germany
*Correspondence:
Christelle M. Andre
christelle.andre@list.lu
Specialty section:
This article was submitted to
Plant Biotechnology,
a section of the journal
Frontiers in Plant Science
Received: 27 October 2015
Accepted: 08 January 2016
Published: 04 February 2016
Citation:
Andre CM, Hausman J-F
and Guerriero G (2016) Cannabis
sativa: The Plant of the Thousand
and One Molecules.
Front. Plant Sci. 7:19.
doi: 10.3389/fpls.2016.00019
INTRODUCTION
The current climatic and economic scenario pushes toward the use of sustainable resources to
reduce our dependence on petrochemicals and to minimize the impact on the environment. Plants
are precious natural resources, because they can supply both phytochemicals and lignocellulosic
biomass. In this review, we focus on hemp (Cannabis sativa L.), since it is a source of bers, oil
and molecules and as such it is an emblematic example of a multi-purpose crop. We treat the
aspects related to the use of hemp biomass and, more extensively, those linked to its wide variety
of phytochemicals.
Andre et al.
Known since the ancient times for its medicinal and textile
uses (Russo et al., 2008; Skoglund et al., 2013), hemp is
currently witnessing a revival, because of its rich repertoire of
phytochemicals, its bers and its agricultural features, namely
quite good resistance to drought and pests, well-developed root
system preventing soil erosion, lower water requirement with
respect to other crops, e.g., cotton. This shows the great versatility
of this ber crop and encourages future studies focused on
both Cannabis (bio)chemistry and genetic engineering. Hemp
varieties producing oil, biomass or even both are currently
cultivated and the availability of the hemp genome sequence
greatly helps molecular studies on this important crop (van
Bakel et al., 2011). In addition, the scientic community is very
much interested in harnessing Cannabis pharmacological power:
for example microorganisms are being engineered to produce
9 -tetrahydrocannabinolic acid (THCA) and cannabidiolic acid
(CBDA) (Taura et al., 2007a; Zirpel et al., 2015).
The nal scope of this review is to discuss the potential
of hemp for industry and to highlight its importance for the
bio-economy. More specically, we: (i) describe the use of
hemp biomass (i.e., the bers), (ii) discuss hemp molecules of
industrial interest (namely cannabinoids, terpenes and phenolic
compounds), (iii) describe the potential of hemp trichomes
as pharma-factories and (iv) discuss the potential of genetic
engineering, by describing the use of plant cell suspension and
hairy root cultures.
Phytocannabinoids
Phytocannabinoids represent a group of C21 or C22 (for the
carboxylated forms) terpenophenolic compounds predominantly
produced in Cannabis. They have also been reported in plants
from the Radula and Helichrysum genus (Appendino et al., 2008)
but our knowledge on non-Cannabis source of cannabinoids is
still in its infancy (Gertsch et al., 2010). More than 90 dierent
cannabinoids have been reported in the literature, although
some of these are breakdown products (ElSohly and Slade, 2005;
Brenneisen, 2007; Radwan et al., 2009; Fischedick et al., 2010) and
they are generally classied into 10 subclasses (Brenneisen, 2007).
In this review, we will focus on the most abundant compounds
found in the drug- and ber-type Cannabis. The predominant
compounds are THCA, CBDA and cannabinolic acid (CBNA),
followed by cannabigerolic acid (CBGA), cannabichromenic
acid (CBCA) and cannabinodiolic acid (CBNDA) (ElSohly and
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FIGURE 1 | Anatomical details of Cannabis stem. (A) Stem of an adult plant (ca 2 months); (B) The stem can be peeled off and shows a lignified core and a
cortex with bast fibers. (C) Longitudinal section of hemp stem stained with toluidine blue showing the cortex with a bundle of bast-fibers (white asterisk) and the core
with xylem vessels (black asterisk).
Slade, 2005). THCA is the major cannabinoid in the drugtype Cannabis, while CBDA predominates in ber-type hemps.
CBCA has been reported to dominate in the cannabinoid
fraction of young plants and to decline with maturation (Meijer
et al., 2009). The phytocannabinoid acids are non-enzymatically
decarboxylated into their corresponding neutral forms, which
occur both within the plant and, to a much larger extent, upon
heating after harvesting (Flores-Sanchez and Verpoorte, 2008).
Phytocannabinoids accumulate in the secretory cavity of the
glandular trichomes, which largely occur in female owers and
in most aerial parts of the plants, as further described in the
next section. They have also been detected in low quantity in
other parts of the plants including the seeds (Ross et al., 2000),
roots (Stout et al., 2012) and the pollen (Ross et al., 2005), in
an extent depending on the drug- or ber-type of Cannabis,
as described in Table 1. More generally, the concentration of
these compounds depends on tissue type (Table 1), age, variety,
growth conditions (nutrition, humidity, light level), harvest
time and storage conditions (Khan et al., 2014). The level of
phytocannabinoids in hempseeds, and thereby of hempseed oil,
should be very low as the kernel contains only trace amount of
THC or CBD (Leizer et al., 2000; Ross et al., 2000). However,
higher THC concentrations are found on the outside surface
of the seed coat, possibly as the result of contamination with
plant leaves or owers (Ross et al., 2000). Recently, signicant
amounts of cannabinoids, and particularly of THC, were found
in ve out of 11 hempseed oil samples available on the Croatian
market, suggesting that both contaminations are due to improper
processing procedures and the illegal use of drug-type hemp
(with a THC + CBN/CBD ratio >1) for nutritional purposes
(Petrovic et al., 2015). Cannabinoids in the leaves have been
shown to decrease with the age and along the stem axis, with
the highest levels observed in the leaves of the uppermost nodes
(Pacico et al., 2008). Cannabinoid contents in the stem are
2000f
1000f
800g
11200g
3000f
1790b
20000f
60300g
22000f
8000e
2000f
3000e
3.4
8.4d
27d
047j
4.2
78d
67
244d
1.67a
CBD
179b
7850
18090j
196
475j
36174
(<2 in
kernel)c
1570d
012
(<0.5 in
kernel)c
329d
1.04a
THC
CBG
1.63a
14.3b
CBN
Molecules
Data are expressed in g g-1 of dry weight. The most recent references have been used, when available. Abbreviations: THC, 9 -tetrahydrocannabinol; CBD, cannabidiol; CBN, cannabinol; CBG, cannabigerol; THCV,
tetrahydrocannabivarin; CBC, cannabichromene. References: a Farag and Kayser, 2015; b Adapted from Stout et al., 2012; c Ross et al., 2000; d Petrovic et al., 2015 (concentration in hempseed oil); e Potter, 2004;
f Pacifico et al., 2008 (growth curve experiment, the maximum concentrations are represented); g Bruci et al., 2012; h Ross et al., 2005; i Fischedick et al., 2010; j Cappelletto et al., 2001, data from stem dust. Commercial
pharmaceutical preparations.
5400i
<600i
CBC
9002200i
(<600)
600i
3240h
THCV
2300i
1700i
1300i
<600i
510h
11200i
100010000i
<600i
1310h
600g
1350h
1300i
79800i
<600i
440h
8590b
6000i
10900g <600i
19000i
190000i
95100g
34000200000i
152000e
76300i
31230h
Drug-type
Drugtype
Drug-type
Drugtype
Fibertype
Drugtype
Fibertype
Fibertype
Root
Drugtype
Fibertype
Seed
Drugtype
Fibertype
Stem
Drugtype
Leaves
Pollen
Fibertype
Flower
Hairy
roots
TABLE 1 | Summary of the concentrations in cannabinoids found in different parts of the hemp plants, in vitro hairy roots, and some commercial medicinal products.
R
Bedrocan
R
Bediol
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FIGURE 2 | Schematic view of the biosynthetic pathways leading to the Cannabis secondary metabolites discussed in this review. Transport of
precursors is represented by dashed arrows, while direct catalytic reactions are depicted by bold arrows. See text for detailed pathways. Abbreviations used: IPP,
isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GPP, geranyl diphosphate; FPP, farnesyl diphosphate; MVA, mevalonate; MEP, methylerythritol
phosphate.
Terpenes
Terpenes form the largest group of phytochemicals, with
more than 100 molecules identied in Cannabis (Rothschild
et al., 2005; Brenneisen, 2007). Terpenes are responsible for
the odor and avor of the dierent Cannabis strains. They
have therefore likely contributed to the selection of Cannabis
narcotic strains under human domestication (Small, 2015).
Terpenes are classied in diverse families according to the
number of repeating units of 5-carbon building blocks (isoprene
Andre et al.
Phenolic Compounds
Phenolic compounds, also known as phenylpropanoids,
constitute one of the most widely distributed group of secondary
metabolites in the plant kingdom. They present more than
10,000 dierent structures, including phenolic acids, such
benzoic and hydroxycinnamic acids, avonoids such as avonols
and avones, stilbenes and lignans (Andre et al., 2010). In
Cannabis, about 20 avonoids have been identied, mainly
belonging to the avone and avonol subclasses (Flores-Sanchez
and Verpoorte, 2008). These include the O-glycoside versions
of the aglycones apigenin, luteolin, kaempferol and quercetin,
as well as cannavin A and cannavin B, which are methylated
isoprenoid avones that are unique to Cannabis (Figure 2)
(Ross et al., 2005). Phenolic amides and lignanamides have also
been described in Cannabis fruits and roots (Sakakibara et al.,
1992; Lesma et al., 2014). The lignanamides belong to the lignan
class of compounds and include cannabisin-like compounds
(of the types A-, B-, C-, D-, E-, F-, and G) and grossamide
(Flores-Sanchez and Verpoorte, 2008). Similar compounds
Andre et al.
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FIGURE 3 | Hemp trichome types. (A) Unicellular non-glandular trichome; (B) cystolythic trichomes; (C) capitate sessile trichome; (D) capitate-stalked trichome;
(E) simple bulbous trichome; (F) complex bulbous trichome. Images kindly provided by Dr. David J. Potter.
FIGURE 4 | Workflow showing the achievements (in green) and potential future approaches (in light blue) to produce cannabinoids in cultures of
Cannabis, as well as other plant hosts.
Andre et al.
CANNABIS BIOTECHNOLOGY:
CHALLENGES AND PROSPECTS
Cannabis is a precious plant with multiple applications, hence
the possibility of engineering it genetically to produce useful
compounds/raw products is highly valuable. In this section of
the review we will: (i) discuss the progress made in Cannabis
in vitro propagation together with the biotechnological prospects
of Cannabis genetic engineering, by highlighting the challenges
and benets, (ii) describe the hairy root culture system as
a tool for the scalable production of cannabinoids and (iii)
discuss the advantages of the Cannabis cell suspension culture
system.
10
Andre et al.
CANNABINOID PRODUCTION IN
HETEROLOGOUS PLANT HOSTS: HOW
IT CAN BE ACHIEVED AND WHAT
SHOULD BE TAKEN INTO ACCOUNT
The expression of genes involved in the cannabinoid biosynthetic
pathway in cell suspension cultures of plants other than
Cannabis represents an interesting alternative for the scalable
11
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12
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involved in biosynthetic pathways of, for instance, lessabundant cannabinoid derivatives. For that purpose,
the combination of metabolomics with genome-based
functional characterizations of gene products would
provide an accelerated path to discovering novel
biosynthetic pathways to specialized metabolites. Indeed,
the functions of numerous genes have been identied and
characterized through the correlation of gene expression
and metabolite accumulation (Sumner et al., 2015).
Classical approaches used focused on the spatial and
temporal distribution of the targeted phytochemicals
and on the plant transcriptome, as inuenced by the
developmental stage and environmental stresses. With respect
to the resurgence of interest in Cannabis phytochemicals
nowadays, the results of such studies will be soon
available.
AUTHOR CONTRIBUTIONS
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Conflict of Interest Statement: The authors declare that the research was
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